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Abstract:

A biologically pure isolate of a selected bacterium derived from
Clostridium autoethanogenum is described which has improved efficiency in
the production of ethanol by anaerobic fermentation of substrates
comprising carbon monoxide. The bacterium can produce ethanol and acetate
at an ethanol to acetate ratio of at least 1.0 and has a productivity of
at least 1.2 g of ethanol/l of fermentation broth per day. The bacterium
is also characterized in that it has substantially no ability to
sporulate.

Claims:

1. A biologically pure isolate of a selected bacterium derived from
Clostridium autoethanogenum, said bacterium characterized by an ability
to produce products comprising ethanol and acetate by anaerobic
fermentation of a substrate comprising CO, wherein the products are
produced at an ethanol to acetate ratio of at least 1.0 and the
productivity of the bacterium is at least 1.2 g of ethanol/1 of
fermentation broth per day.

2. The selected bacterium of claim 1 where the ethanol:acetate ratio is
at least 1.2.

3. The selected bacterium of claim 2 where the bacterium's productivity
of ethanol is at least 2.0 g ethanol/L fermentation broth/day.

4. The selected bacterium of claim 1 where the selected bacterium has
substantially no ability to sporulate and at least one of the other
defining characteristics: (i) an ability to grow in minimal media in the
presence or absence of yeast extract; (ii) an ability to grow more
rapidly, to produce a higher ratio of ethanol to acetate, and/or to
produce a higher concentration of ethanol, in a media in which yeast
extract is not present compared to a media in which yeast extract is
present; and (iii) non-motile.

5. The selected bacterium of claim 4 wherein the other defining
characteristics are: (i) the ability to grow in minimal media in the
presence or absence of yeast extract; and (ii) the ability to produce a
higher concentration of ethanol in a media in which yeast extract is not
present compared to a media in which yeast extract is present.

6. The selected bacterium of claim 4 wherein the other defining
characteristics are: (i) the ability to produce a higher ratio of ethanol
to acetate in a media in which yeast extract is not present compared to a
media in which yeast extract is present: and (ii) non-motile.

7. The selected bacterium of claim 4 wherein the other defining
characteristics are the ability to grow more rapidly, to produce a higher
ratio of ethanol to acetate and to produce a higher concentration of
ethanol in a media in which yeast extract is not present compared to a
media in which yeast extract is present.

8. The selected bacterium of claim 4 wherein the other defining
characteristics are: (i) an ability to grow in minimal media in the
presence or absence of yeast extract; (ii) an ability to grow more
rapidly, to produce a higher ratio of ethanol to acetate, and to produce
a higher concentration of ethanol, in a media in which yeast extract is
not present compared to a media in which yeast extract is present; and
(iii) non-motile.

9. A biologically pure isolate of a selected bacterium derived from
Clostridium autoethanogenum, said bacterium having substantially no
ability to sporulate and at least one of the other defining
characteristics: (i) an ability to grow in minimal media in the presence
or absence of yeast extract; (ii) an ability to grow more rapidly, to
produce a higher ratio of ethanol to acetate, and/or to produce a higher
concentration of ethanol, in a media in which yeast extract is not
present compared to a media in which yeast extract is present; and (iii)
non-motile.

10. The selected bacterium of claim 9 where the bacterium ferments a
substrate comprising CO to products comprising ethanol and acetate and
the ratio of ethanol to acetate is at least 1.0 and where the bacterium's
productivity of ethanol is at least 1.2 g ethanol/L fermentation
broth/day.

11. The selected bacterium of claim 10 where the ethanol:acetate ratio is
at least 1.2.

12. The selected bacterium of claim 10 where the bacterium's productivity
of ethanol is at least 2.0 g ethanol/L fermentation broth/day.

13. The selected bacterium of claim 9 wherein the other defining
characteristics are: (i) the ability to grow in minimal media in the
presence or absence of yeast extract; and (ii) the ability to produce a
higher concentration of ethanol in a media in which yeast extract is not
present compared to a media in which yeast extract is present.

14. The selected bacterium of claim 9 wherein the other defining
characteristics are: (i) the ability to produce a higher ratio of ethanol
to acetate in a media in which yeast extract is not present compared to a
media in which yeast extract is present: and (ii) non-motile.

15. The selected bacterium of claim 9 wherein the other defining
characteristics are the ability to grow more rapidly, to produce a higher
ratio of ethanol to acetate and to produce a higher concentration of
ethanol in a media in which yeast extract is not present compared to a
media in which yeast extract is present.

16. The selected bacterium of claim 9 wherein the other defining
characteristics are: (i) an ability to grow in minimal media in the
presence or absence of yeast extract; (ii) an ability to grow more
rapidly, to produce a higher ratio of ethanol to acetate, and to produce
a higher concentration of ethanol, in a media in which yeast extract is
not present compared to a media in which yeast extract is present; and
(iii) non-motile.

Description:

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. application Ser.
No. 12/742,149 filed on Aug. 5, 2010 which in turn is a National Stage of
International Application No. PCT/NZ2008/000305, filed on Nov. 13, 2008,
which claims priority to U.S. Provisional Application 60/987,755 filed on
Nov. 13, 2007; all of the contents of all said prior applications are
incorporated by reference in their entirety.

FIELD OF THE INVENTION

[0002] This invention relates generally to the field of microbial
fermentation of gases. It more particularly relates to a novel class of
bacteria with improved efficiency in the production of ethanol by
anaerobic fermentation of substrates containing carbon monoxide (CO).

BACKGROUND OF THE INVENTION

[0003] Ethanol is rapidly becoming a major hydrogen-rich liquid transport
fuel around the world. Worldwide consumption of ethanol in 2005 was an
estimated 12.2 billion gallons. The global market for the fuel ethanol
industry has also been predicted to grow sharply in future, due to an
increased interest in ethanol in Europe, Japan, the USA, and several
developing nations.

[0004] For example, in the USA, ethanol is used to produce E10, a 10%
mixture of ethanol in gasoline. In E10 blends the ethanol component acts
as an oxygenating agent, improving the efficiency of combustion and
reducing the production of air pollutants. In Brazil, ethanol satisfies
approximately 30% of the transport fuel demand, as both an oxygenating
agent blended in gasoline, and as a pure fuel in its own right. Also, in
Europe, environmental concerns surrounding the consequences of Green
House Gas (GHG) emissions have been the stimulus for the European Union
(EU) to set member nations a mandated target for the consumption of
sustainable transport fuels such as biomass derived ethanol.

[0005] The vast majority of fuel ethanol is produced via traditional
yeast-based fermentation processes that use crop derived carbohydrates,
such as sucrose extracted from sugarcane or starch extracted from grain
crops, as the main carbon source. However, the cost of these carbohydrate
feed stocks is influenced by their value as human food or animal feed,
while the cultivation of starch or sucrose-producing crops for ethanol
production is not economically sustainable in all geographies. Therefore,
it is of interest to develop technologies to convert lower cost and/or
more abundant carbon resources into fuel ethanol.

[0006] CO is a major, free, energy-rich by-product of the incomplete
combustion of organic materials such as coal or oil and oil derived
products. For example, the steel industry in Australia is reported to
produce and release into the atmosphere over 500,000 tonnes of CO
annually.

[0007] Catalytic processes may be used to convert gases consisting
primarily of CO and/or CO and hydrogen (H2) into a variety of fuels and
chemicals. Micro-organisms may also be used to convert these gases into
fuels and chemicals.

[0008] The ability of micro-organisms to grow on CO as a sole carbon
source was first discovered in 1903. This was later determined to be a
property of organisms that use the acetyl coenzyme A (acetyl CoA)
biochemical pathway of autotrophic growth (also known as the
Woods-Ljungdahl pathway and the carbon monoxide dehydrogenase/acetyl CoA
synthase (CODH/ACS) pathway). A large number of anaerobic organisms
including carboxydotrophic, photosynthetic, methanogenic and acetogenic
organisms have been shown to metabolize CO to various end products,
namely CO2, H2, methane, n-butanol, acetate and ethanol. While using CO
as the sole carbon source, all such organisms produce at least two of
these end products.

[0009] Anaerobic bacteria, such as those from the genus Clostridium, have
been demonstrated to produce ethanol from CO, CO2 and H2 via the acetyl
CoA biochemical pathway. For example, various strains of Clostridium
ljungdahlii that produce ethanol from gases are described in WO 00/68407,
EP 117309, U.S. Pat. Nos. 5,173,429, 5,593,886, and 6,368,819, WO
98/00558 and WO 02/08438. The bacterium Clostridium autoethanogenum sp is
also known to produce ethanol from gases (Abrini et al., Archives of
Microbiology 161, pp 345-351 (1994)).

[0010] However, ethanol production by micro-organisms by fermentation of
gases is always associated with co-production of acetate and/or acetic
acid. As some of the available carbon is converted into acetate/acetic
acid rather than ethanol, the efficiency of production of ethanol using
such fermentation processes may be less than desirable. Also, unless the
acetate/acetic acid by-product can be used for some other purpose, it may
pose a waste disposal problem. Acetate/acetic acid is converted to
methane by micro-organisms and therefore has the potential to contribute
to GHG emissions.

[0011] Microbial fermentation of CO in the presence of H2 can lead to
substantially complete carbon transfer into an alcohol. However, in the
absence of sufficient H2, some of the CO is converted into alcohol, while
a significant portion is converted to CO2 as shown in the following
equations:

6CO+3H2O→C2H5OH+4CO2

12H2+4CO2→2C2H5OH+6H2O

[0012] The production of CO2 represents inefficiency in overall
carbon capture and if released, also has the potential to contribute to
Green House Gas emissions.

[0013] WO2007/117157 describes a process that produces alcohols,
particularly ethanol, by anaerobic fermentation of gases containing
carbon monoxide. Acetate produced as a by-product of the fermentation
process is converted into hydrogen gas and carbon dioxide gas, either or
both of which may be used in the anaerobic fermentation process.

[0014] WO2008/115080 describes a process for the production of alcohol(s)
in multiple fermentation stages. By-products produced as a result of
anaerobic fermentation of gas(es) in a first bioreactor can be used to
produce products in a second bioreactor. Furthermore, by-products of the
second fermentation stage can be recycled to the first bioreactor to
produce products.

[0015] It would thus be beneficial to provide micro-organisms that are
capable of fermentation of such gases to ethanol at increased efficiency,
that is micro-organisms capable of producing more ethanol, and/or a
greater ratio of ethanol to acetate from the same substrate, than do
micro-organisms of the prior art.

[0016] In addition, in prior art methods of bacterial fermentation of
CO-containing gases to ethanol that produce high levels of ethanol and/or
a high ethanol to acetate ratio, the gaseous substrate used typically
comprises about 30-65% CO by volume and about 20-30% H2 by volume
(WO 00/68407).

[0017] CO-containing waste gases, that are potential substrates for
microbial fermentation to produce ethanol, may contain either higher
levels of CO and lower levels of H2 or both. It would therefore be
beneficial to have available bacterial strains that can perform efficient
fermentation of CO-containing gas with greater than 65% CO by volume and
or less than 20% H2 by volume into ethanol, for example.

[0018] It is an object of the present invention to provide a new class of
bacteria which overcomes one or more of the limitations of the prior art
in the conversion of gaseous sources containing CO into ethanol.

SUMMARY OF THE INVENTION

[0019] In a first aspect the invention provides a biologically pure
isolate of a selected bacterium capable of producing products including
ethanol and optionally acetate, by anaerobic fermentation of a substrate
comprising CO, wherein the products are produced at an ethanol to acetate
ratio of at least 1.0.

[0020] In another aspect the invention provides a biologically pure
isolate of a selected bacterium capable of producing ethanol and acetate
by anaerobic fermentation in an aqueous culture medium supplied with a
substrate comprising CO, particularly a gaseous substrate containing CO,
comprising:

[0021] (a) greater than about 65% CO by volume

[0022] (b) less than about 20% H2 by volume, or

[0023] (c) greater than about 65% CO and less than about 20% H2 by
volume,

at an ethanol to acetate ratio of at least about 1.0.

[0024] In one particular embodiment the ethanol to acetate ratio is at
least about 1.1, more preferably at least about 1.2, more preferably at
least about 1.3 and most preferably at least about 1.4.

[0025] In a further embodiment the bacterium is capable of producing the
ethanol at a concentration of at least about 2.0 g ethanol per litre of
fermentation broth.

[0026] In particular embodiments the concentration is at least about 2.1 g
ethanol per litre of fermentation broth, at least about 2.2 g ethanol per
litre of fermentation broth, at least about 2.3 g ethanol per litre of
fermentation broth, at least about 2.4 g ethanol per litre of
fermentation broth, at least about 2.5 g ethanol per litre of
fermentation broth, at least about 2.6 g ethanol per litre of
fermentation broth, at least about 2.7 g ethanol per litre of
fermentation broth at least about 2.8 g ethanol per litre of fermentation
broth, at least about 3.0 g ethanol per litre of fermentation broth, at
least about 3.2 g ethanol per litre of fermentation broth, or at least
about 3.4 g ethanol per litre of fermentation broth.

[0027] In particular embodiments the productivity of the bacterium is at
least about 1.2 g of ethanol/L of fermentation broth/day, at least about
1.6 g/L/day, at least about 1.8 g/L/day or at least 2.0 g/L/day.

[0028] In certain embodiments, the specific ethanol productivity of the
bacterium is at least about 0.7 g/L/gram bacterial cells/day, at least
about 0.9 g/L/gram bacterial cells/day, at least about 1.1 g/L/gram
bacterial cells/day, or at least about 1.3 g/L/gram bacterial cells/day.

[0029] In another aspect the invention provides a biologically pure
isolate of a selected bacterium capable of producing products including
alcohol and optionally acetate, by anaerobic fermentation of a substrate
comprising CO, wherein the productivity of the bacterium is at least
about 1.2 g of ethanol/L of fermentation broth/day.

[0030] In a further aspect the invention provides a biologically pure
isolate of a selected bacterium capable of producing ethanol by anaerobic
fermentation in an aqueous culture medium supplied with a substrate
containing CO, particularly a gaseous substrate containing CO,
comprising:

[0031] (a) greater than about 65% CO by volume

[0032] (b) less than about 20% H2 by volume, or

[0033] (c) greater than about 65% CO and less than about 20% H2 by
volume,

at an ethanol concentration of at least about 2.0 g ethanol per litre of
fermentation broth.

[0034] In particular embodiments the concentration is at least about 2.1 g
ethanol per litre of fermentation broth, at least about 2.2 g ethanol per
litre of fermentation broth, at least about 2.3 g ethanol per litre of
fermentation broth, at least about 2.4 g ethanol per litre of
fermentation broth, at least about 2.5 g ethanol per litre of
fermentation broth, at least about 2.6 g ethanol per litre of
fermentation broth, at least about 2.7 g ethanol per litre of
fermentation broth at least about 2.8 g ethanol per litre of fermentation
broth, at least about 3.0 g ethanol per litre of fermentation broth, at
least about 3.2 g ethanol per litre of fermentation broth, or at least
about 3.4 g ethanol per litre of fermentation broth.

[0035] In particular embodiments the productivity of the bacterium is at
least about 1.2 g of ethanol/L of fermentation broth/day, at least about
1.6 g/L/day, at least about 1.8 g/L/day or at least 2.0 g/L/day.

[0036] In certain embodiments, the specific ethanol productivity of the
bacterium is at least about 0.7 g/L/gram bacterial cells/day, at least
about 0.9 g/L/gram bacterial cells/day, at least about 1.1 g/L/gram
bacterial cells/day, or at least about 1.3 g/L/gram bacterial cells/day.

[0037] In one embodiment acetate is produced as a by-product of the
fermentation.

[0038] In a particular embodiment the ethanol is produced at an ethanol to
acetate ratio of at least about 1.0. In particular embodiments the
ethanol to acetate ratio is at least about 1.1, at least about 1.2, at
least about 1.3 or more particularly at least about 1.4.

[0039] In another aspect, the invention provides an acetogenic bacterium
wherein the bacterium has one or more of the following defining
characteristics:

[0040] an ability to grow in minimal medium in the presence or absence of
yeast extract;

[0041] an ability to grow more rapidly, to produce a higher ratio of
ethanol to acetate, and/or to produce a higher concentration of ethanol,
in a medium in which yeast extract is not present compared to a medium in
which yeast extract is present;

[0042] little or no ability to sporulate;

[0043] Gram positive;

[0044] rod shaped;

[0045] Non-motile.

[0046] In one embodiment the bacteria are additionally capable of
producing ethanol by anaerobic fermentation in an aqueous culture medium
supplied with a CO-containing substrate comprising:

[0047] (a) greater than about 65% CO by volume,

[0048] (b) less than about 20% H2 by volume, or

[0049] (c) greater than about 65% CO and less than about 20% H2 by
volume,

at an ethanol concentration of at least about 2.0 g ethanol per litre of
fermentation broth and/or at an ethanol to acetate ratio of at least
about 1.0.

[0050] In particular embodiments the ethanol to acetate ratio is at least
about 1.1, at least about 1.2, at least about 1.3 or more particularly at
least about 1.4.

[0051] In particular embodiments the concentration of ethanol produced is
at least about 2.1 g ethanol per litre of fermentation broth, at least
about 2.2 g ethanol per litre of fermentation broth, at least about 2.3 g
ethanol per litre of fermentation broth, at least about 2.4 g ethanol per
litre of fermentation broth, at least about 2.5 g ethanol per litre of
fermentation broth, at least about 2.6 g ethanol per litre of
fermentation broth, at least about 2.7 g ethanol per litre of
fermentation broth at least about 2.8 g ethanol per litre of fermentation
broth, at least about 3.0 g ethanol per litre of fermentation broth, at
least about 3.2 g ethanol per litre of fermentation broth, or at least
about 3.4 g ethanol per litre of fermentation broth.

[0052] In particular embodiments the productivity of the bacterium is at
least about 1.2 g of ethanol/L of fermentation broth/day, at least about
1.6 g/L/day, at least about 1.8 g/L/day or at least 2.0 g/L/day.

[0053] In certain embodiments, the specific ethanol productivity of the
bacterium is at least about 0.7 g/L/gram bacterial cells/day, at least
about 0.9 g/L/gram bacterial cells/day, at least about 1.1 g/L/gram
bacterial cells/day, or at least about 1.3 g/L/gram bacterial cells/day.

[0054] In one embodiment, the bacteria of the invention are derived from
Clostridium autoethanogenum.

[0055] In a particular embodiment, the bacteria have two or more and most
preferably all of the above defining characteristics.

[0056] In a particular embodiment the bacterium has the defining
characteristics of Clostridium autoethanogenum strain LBS1560 deposited
at DSMZ under the accession number DSM 19630. In one embodiment the
bacterium is Clostridium autoethanogenum strain LBS1560 deposited at DSMZ
under the accession number DSM 19630.

[0057] In a further aspect the invention provides a biologically pure
isolate of Clostridium autoethanogenum strain LBS1560 deposited at DSMZ
under the accession number DSM 19630.

[0058] In one embodiment the substrate comprises at least about 70% CO by
volume, at least about 75% CO by volume, at least about 80% CO by volume,
at least about 85% CO by volume, at least about 90% CO by volume or at
least about 95% CO by volume.

[0059] In a further embodiment the substrate comprises less than about 20%
H2 by volume. In particular embodiments the substrate comprises less
than about 15% H2 by volume, less than about 10% H2 by volume,
less than about 5% H2 by volume, less than about 4% H2 by
volume, less than about 3% H2 by volume, less than about 2% H2
by volume, less than about 1% H2 by volume, or substantially no
H2.

[0060] In a further embodiment the substrate comprises less than or equal
to about 20% CO2 by volume. In particular embodiments the substrate
comprises less than or equal to about 15% CO2 by volume, less than
or equal to about 10% CO2 by volume, or less than or equal to about
5% CO2 by volume.

[0061] In particular embodiments the substrate comprises at least about
85% CO by volume and at most about 15% CO2 by volume, at least about
90% CO and at most about 10% CO2, or about 95% CO by volume and
about 5% CO2 by volume.

[0062] In certain embodiments the aqueous culture medium is a minimal
anaerobic microbial growth medium selected from but not limited to LM23
or LM33 as herein defined.

[0063] In one embodiment, the medium is not supplemented with yeast
extract.

[0064] In a further aspect, the invention provides a method for the
production of one or more alcohols from a substrate containing CO, the
method comprising maintaining a culture of one or more of the bacterial
isolates of the invention in the presence of the substrate, and the
anaerobic fermentation of the substrate to one or more alcohols by the
one or more bacterial isolate.

[0065] In another aspect, the invention provides a method for the
production of one or more alcohols comprising fermenting a substrate
containing CO using one or more of the bacteria as herein before
described.

[0066] In one embodiment the method comprises the steps of: [0067] (a)
providing a substrate containing CO to a bioreactor containing a culture
of a bacterium as hereinbefore described; and [0068] (b) anaerobically
fermenting the culture in the bioreactor to produce one or more alcohols.

[0069] In a further aspect, the invention provides a method for reducing
the total atmospheric carbon emissions from an industrial process, the
method comprising: [0070] (a) capturing CO-containing gas produced as a
result of the industrial process, before the gas is released into the
atmosphere; [0071] (b) the anaerobic fermentation of the CO-containing
gas to produce one or more alcohols by a culture containing one or more
bacterial isolates of the invention.

[0072] In certain embodiments of the method aspects, acetate is produced
as a by-product of the fermentation. Preferably the one or more alcohols
produced includes ethanol.

[0073] In particular embodiments of the method aspects, the bacterium or
isolate is maintained in an aqueous culture medium.

[0074] In particular embodiments of the method aspects, the fermentation
of the substrate takes place in a bioreactor.

[0075] In certain embodiments, the substrate contains less than about 15%
H2 by volume, such as less than about 10% H2, such as less than
about 5% H2.

[0076] In certain embodiments, the substrate comprises greater than about
65% CO by volume, preferably about 70% CO to about 95% CO by volume.

[0077] In one embodiment the substrate comprises at least about 70% CO by
volume. In a particular embodiment the substrate comprises at least about
80% CO by volume, at least about 85% CO by volume, at least about 90% CO
by volume or at least about 95% CO by volume.

[0078] In one embodiment the substrate comprises less than about 20%
H2 by volume. In particular embodiments the substrate comprises less
than about 15% H2 by volume, less than about 10% H2 by volume,
less than about 5% H2 by volume, less than about 4% H2 by
volume, less than about 3% H2 by volume, less than about 2% H2
by volume, less than about 1% H2 by volume, or substantially no
H2.

[0079] In one embodiment the substrate comprises less than or equal to
about 20% CO2 by volume. In particular embodiments the substrate
comprises less than or equal to about 15% CO2 by volume, less than
or equal to about 10% CO2 by volume, or less than or equal to about
5% CO2 by volume.

[0080] In certain embodiments the substrate comprises at least about 85%
CO by volume and at most about 15% CO2 by volume, at least about 90%
CO and at most about 10% CO2, or about 95% CO by volume and about 5%
CO2 by volume.

[0084] In one embodiment, the gaseous substrate may comprise a gas
obtained from a steel mill.

[0085] In another embodiment, the gaseous substrate may comprise
automobile exhaust fumes.

[0086] In certain embodiments of the method aspects the alcohol is
recovered from the fermentation broth, the fermentation broth being the
aqueous culture medium comprising bacterial cells and the alcohol.

[0087] In certain embodiments acetate is produced as a by-product of the
fermentation.

[0088] In a further embodiment the alcohol and the acetate are recovered
from the broth.

[0089] In another aspect, the invention provides a method of selection of
one or more micro-organisms which produce one or more acids, the method
comprising: Culturing the micro-organisms in a nutrient medium in a
bioreactor; Adding fresh medium at a pH higher than the nutrient medium,
such that the nutrient medium is maintained at a substantially constant
pH; and, Removing at least a portion of the nutrient medium and
micro-organisms, such that the medium in the bioreactor is maintained at
a substantially constant volume.

[0090] In a particular embodiment, the method is for the selection of fast
growing micro-organisms. In one embodiment the one or more acids includes
acetate.

[0091] In another aspect the invention provides a biologically pure
isolate of a bacterium produced by the method of selection. In one
embodiment, the isolate has little or no ability to sporulate.

[0092] Although the invention is broadly as defined above, it is not
limited thereto and also includes embodiments of which the following
description provides examples.

BRIEF DESCRIPTION OF THE DRAWINGS

[0093] The invention will now be described in detail with reference to the
accompanying Figures in which:

[0094] FIG. 1: is a schematic representation of a system adapted to select
for rapid microbial growth

[0096] In broad terms, in one aspect the present invention relates to a
novel selected bacterium and a biologically pure isolate of the selected
bacterium with increased efficiency in an anaerobic fermentation process.
In one aspect the bacterium is capable of producing an alcohol,
preferably ethanol, from a substrate comprising:

[0097] (a) greater than about 65% CO by volume

[0098] (b) less than about 20% H2 by volume, or

[0099] (c) greater than about 65% CO and less than about 20% H2 by
volume.

[0100] In a further aspect, the invention relates to a process for
producing an alcohol, preferably ethanol, by anaerobic fermentation of a
CO-containing substrate by the bacteria of the invention.

DEFINITIONS

[0101] Unless otherwise defined, the following terms as used throughout
this specification are defined as follows:

[0102] A "substrate containing CO" and like terms should be understood to
include any substrate in which carbon monoxide is available to bacteria
for growth and/or fermentation, for example. In particular embodiments of
the invention the "substrate containing CO" is gaseous. Such substrates
may be referred to herein as "gaseous substrates containing CO" and the
like.

[0103] In the description which follows, embodiments of the invention are
described in terms of delivering and fermenting a "gaseous substrate
containing CO". However, it should be appreciated that the gaseous
substrate may be provided in alternative forms. For example, the gaseous
substrate containing CO may be provided dissolved in a liquid.
Essentially, a liquid is saturated with a carbon monoxide containing gas
and then that liquid is added to the bioreactor. This may be achieved
using standard methodology. By way of example, a microbubble dispersion
generator (Hensirisak et. al. Scale-up of microbubble dispersion
generator for aerobic fermentation; Applied Biochemistry and
Biotechnology Volume 101, Number 3/October, 2002) could be used. By way
of further example, the gaseous substrate containing CO may be adsorbed
onto a solid support. Such alternative methods are encompassed by use of
the term "substrate containing CO".

[0104] The terms "increasing the efficiency", "increased efficiency" and
the like, when used in relation to a fermentation process, include, but
are not limited to, increasing one or more of: the rate of growth of
micro-organisms catalysing the fermentation, the volume of desired
product (such as alcohols) produced per volume of substrate (such as CO)
consumed, the concentration of the desired product (such as alcohols)
produced in the culture medium, the rate of production or level of
production of the desired product, and the relative proportion of the
desired product produced compared with other by-products of the
fermentation.

[0105] The term "acetate" includes both acetate salt alone and a mixture
of molecular or free acetic acid and acetate salt, such as the mixture of
acetate salt and free acetic acid present in a fermentation broth as
described herein. The ratio of molecular acetic acid to acetate in the
fermentation broth is dependent upon the pH of the system.

[0106] The term "bioreactor" includes a fermentation device consisting of
one or more vessels and/or towers or piping arrangement, which includes
the Continuous Stirred Tank Reactor (CSTR), Immobilized Cell Reactor
(ICR), Trickle Bed Reactor (TBR), Bubble Column, Gas Lift Fermenter,
Static Mixer, or other vessel or other device suitable for gas-liquid
contact.

[0107] Bacteria of the invention, or cultures or isolates thereof, may be
described to be in an "isolated" or "biologically pure" form. These terms
are intended to mean that the bacteria have been separated from an
environment or one or more constituents, cellular or otherwise, which
they may be associated with if found in nature or otherwise. The terms
"isolated" or "biologically pure" should not be taken to indicate the
extent to which the bacteria have been purified. However, in one
embodiment the isolates or cultures of the bacteria contain a
predominance of the bacteria of the invention.

[0108] The term "selected bacterium" refers to a bacterium produced
through a dedicated program of selection and propagation of microbial
cultures as described in example 1 below.

[0109] The invention provides a biologically pure isolate of a bacterium
capable of producing ethanol and acetate by anaerobic fermentation in an
aqueous culture medium supplied with a gaseous CO-containing substrate
comprising:

[0110] (a) greater than about 65% CO by volume

[0111] (b) less than about 20% H2 by volume, or

[0112] (c) greater than about 65% CO and less than about 20% H2 by
volume,

at an ethanol to acetate ratio of at least about 1.0. In one embodiment,
the bacterium is derived from C. autoethanogenum as described elsewhere
herein.

[0113] In certain embodiments the ethanol to acetate ratio is at least
about 1.1, or at least about 1.2, or at least about 1.3 or at least about
1.4. It should be pointed out that the ratio of products produced
(ethanol:acetate) and the ethanol productivity described below are the
result of the intrinsic properties of the bacterium and not merely the
result from adjusting the fermentation parameters.

[0114] In further embodiments the bacterium is capable of producing
ethanol at a concentration of at least about 2.1 g ethanol per litre of
fermentation broth, at least about 2.2 g ethanol per litre of
fermentation broth, at least about 2.3 g ethanol per litre of
fermentation broth, at least about 2.4 g ethanol per litre of
fermentation broth, at least about 2.5 g ethanol per litre of
fermentation broth, at least about 2.6 g ethanol per litre of
fermentation broth, at least about 2.7 g ethanol per litre of
fermentation broth at least about 2.8 g ethanol per litre of fermentation
broth, at least about 3.0 g ethanol per litre of fermentation broth, at
least about 3.2 g ethanol per litre of fermentation broth, or at least
about 3.4 g ethanol per litre of fermentation broth.

[0115] Ethanol productivity is the volumetric productivity of ethanol,
calculated as the ratio of the ethanol concentration and the time
required to produce that concentration in batch systems. Productivity can
also be calculated for microbial fermentation in continuous systems. In
particular embodiments of the invention, the productivity of the bacteria
is at least 1.2 g ethanol/L of fermentation broth/day, or at least 1.6
g/L/day or at least 1.8 g/L/day or at least 2.0 g/L/day.

[0116] The specific productivity of a microbial culture depends on the
proportion of live active microorganism within a microbial culture. In
certain embodiments of the present invention, the specific ethanol
productivity is at least 0.7 g/L/gram bacterial cells/day, or at least
0.9 g/L/gram bacterial cells/day, or at least 1.1 g/L/gram bacterial
cells/day, or at least 1.3 g/L/gram bacterial cells/day.

[0117] The invention also provides a biologically pure isolate of a
bacterium capable of producing ethanol by anaerobic fermentation in an
aqueous culture medium supplied with a gaseous CO-containing substrate
comprising:

[0118] (a) greater than about 65% CO by volume

[0119] (b) less than about 20% H2 by volume, or

[0120] (c) greater than about 65% CO and less than about 20% H2 by
volume,

at an ethanol concentration of at least 2.0 g ethanol per litre of
fermentation broth. In one embodiment, the bacterium is derived from C.
autoethanogenum as described elsewhere herein.

[0121] In further embodiments the bacterium is capable of producing
ethanol at a concentration of at least about 2.1 g ethanol per litre of
fermentation broth, at least about 2.2 g ethanol per litre of
fermentation broth, at least about 2.3 g ethanol per litre of
fermentation broth, at least about 2.4 g ethanol per litre of
fermentation broth, at least about 2.5 g ethanol per litre of
fermentation broth, at least about 2.6 g ethanol per litre of
fermentation broth, at least about 2.7 g ethanol per litre of
fermentation broth at least about 2.8 g ethanol per litre of fermentation
broth, at least about 3.0 g ethanol per litre of fermentation broth, at
least about 3.2 g ethanol per litre of fermentation broth, or at least
about 3.4 g ethanol per litre of fermentation broth.

[0122] In particular embodiments of the invention, the productivity of the
bacteria is at least 1.2 g ethanol/L of fermentation broth/day, or at
least 1.6 g/L/day or at least 1.8 g/L/day or at least 2.0 g/L/day.

[0123] In certain embodiments of the present invention, the specific
ethanol productivity is at least 0.7 g/L/gram bacterial cells/day, or at
least 0.9 g/L/gram bacterial cells/day, or at least 1.1 g/L/gram
bacterial cells/day, or at least 1.3 g/L/gram bacterial cells/day.

[0124] Typically acetate is produced as a by-product of the fermentation.
In one embodiment the ethanol is produced at an ethanol to acetate ratio
of at least about 1.0. In particular embodiments the ethanol to acetate
ratio is at least about 1.1, or at least about 1.2, or at least about 1.3
or at least about 1.4.

[0125] The invention also provides acetogenic bacteria having one or more
of the following defining characteristics as observed under the
experimental conditions described herein after: an ability to grow in
minimal medium in the presence or absence of yeast extract; an ability to
grow more rapidly, to produce a higher ratio of ethanol to acetate,
and/or to produce a higher concentration of ethanol, in a medium in which
yeast extract is not present compared to a medium in which yeast extract
is present; little or no ability to sporulate; Gram positive; rod shaped;
Non-motile.

[0126] In one embodiment the bacteria have substantially no ability to
sporulate. In one embodiment substantially none of the bacterial
population exhibit spores under the conditions described herein after.

[0127] In one embodiment the acetogenic bacteria are additionally capable
of producing ethanol by anaerobic fermentation in an aqueous culture
medium supplied with a CO-containing substrate comprising: greater than
about 65% CO by volume; less than about 20% H2 by volume; or,
greater than about 65% CO and less than about 20% H2 by volume; at
an ethanol concentration of at least about 2.0 g ethanol per litre of
fermentation broth and/or at an ethanol to acetate ratio of at least
about 1.0.

[0128] The bacteria of the invention can be derived from Clostridium
autoethanogenum.

[0129] The observation that bacteria of certain embodiments of the
invention have little to no ability to sporulate is surprising. This
provides an unexpected benefit over other strains of Clostridia including
Clostridium autoethanogenum described in the prior art. For example
Abrini et al (see abstract) describes a Clostridium autoethanogenum which
is spore forming Sporulation is a stagnant phase of limited activity.
Reducing or ameliorating the ability to form spores has a number of
advantages. For example, a single bacterial cell can only divide and
produce metabolites (such as acetate and/or ethanol) while in a non
sporulated condition. Accordingly, the time scale for division and
metabolite production can be extended where bacteria do not sporulate.
The lack of an ability to sporulate may also provide additional control
over an entire culture, wherein the whole live population may be adapted
to promote growth and/or metabolite production for extended periods.
Therefore, use of bacteria of the present invention may increase the
overall efficiency of a fermentation process for producing products such
as acetate and/or ethanol.

[0130] In certain embodiments of the invention, the bacteria have two or
more and more preferably all of the above mentioned characteristics. In
some embodiments of the invention the bacteria have the defining
characteristics of Clostridium autoethanogenum strain LBS1560 deposited
at DSMZ, Germany, in accordance with the Budapest Treaty, on 19 Oct.
2007, and allocated the accession number DSM 19630. In a particular
embodiment, the bacterium is Clostridium autoethanogenum strain LBS1560,
DSM 19630.

[0131] The invention also relates to bacteria derived from the bacteria of
the invention.

[0132] In certain embodiments the bacteria of the invention are able to
produce the concentrations of ethanol, and ethanol to acetate ratios
discussed above, at elevated levels of CO in the gaseous substrate. The
gaseous substrate may comprise at least about 70% CO by volume. In
certain embodiments the gaseous substrate comprises at least about 80% CO
by volume, or at least about 85% CO by volume, or at least about 90% CO
by volume or at least about 95% CO by volume.

[0133] Similarly the discussed ethanol concentrations, and ethanol to
acetate ratios, are achievable in certain embodiments at low to
non-existent levels of H2 in the gaseous substrate. The gaseous
substrate may comprise less than about 20% H2 by volume. In
particular embodiments the gaseous substrate comprises less than about
15% H2 by volume, or the gaseous substrate comprises less than about
10% H2 by volume, or the gaseous substrate comprises less than about
5% H2 by volume, or the gaseous substrate comprises less than about
4% H2 by volume, or the gaseous substrate comprises less than about
3% H2 by volume, or the gaseous substrate comprises less than about
2% H2 by volume, or the gaseous substrate comprises less than about
1% H2 by volume, or the gaseous substrate comprises no H2.

[0134] In certain embodiments, the bacteria of the invention can also
produce ethanol concentrations, and ethanol to acetate ratios when
supplied with gaseous substrate comprising relatively little CO2. In
one embodiment the gaseous substrate comprises less than or equal to
about 20% CO2 by volume. In certain embodiments the gaseous
substrate comprises less than or equal to about 15% CO2 by volume,
or less than or equal to about 10% CO2 by volume, or less than or
equal to about 5% CO2 by volume.

[0135] In certain embodiments the gaseous substrate comprises about 85% CO
by volume and about 15% CO2 by volume, or the gaseous substrate
comprises at least about 90% CO and at most about 10% CO2, or the
gaseous substrate comprises about 95% CO by volume and about 5% CO2
by volume.

[0136] In certain embodiments the culture is maintained in an aqueous
culture medium. Preferably the aqueous culture medium is a minimal
anaerobic microbial growth medium. Suitable medium are known in the art
and described for example in U.S. Pat. Nos. 5,173,429 and 5,593,886 and
WO 02/08438, and in Klasson et al [(1992). Bioconversion of Synthesis Gas
into Liquid or Gaseous Fuels. Enz. Microb. Technol. 14:602-608.],
Najafpour and Younesi [(2006). Ethanol and acetate synthesis from waste
gas using batch culture of Clostridium ljungdahlii. Enzyme and Microbial
Technology, Volume 38, Issues 1-2, p. 223-228] and Lewis et al [(2002).
Making the connection-conversion of biomass-generated producer gas to
ethanol. Abst. Bioenergy, p. 2091-2094.]. In particular embodiments of
the invention, the minimal anaerobic microbial growth medium is LM23 or
LM33 as herein defined.

[0137] In certain embodiments the medium is supplemented with additional
components, such as but not limited to amino acids and trypticase.
Preferably the medium is not supplemented with additional components.

[0138] In certain embodiments the medium may be supplemented with yeast
extract. In certain embodiments the culture grows more rapidly when the
medium is not supplemented with yeast extract, than when the medium is
supplemented with yeast extract. In a further embodiment the ethanol to
acetate ratio produced is higher when the medium is not supplemented with
yeast extract, than when the medium is supplemented with yeast extract.
In a further embodiment the concentration of ethanol produced per litre
of culture medium is higher when the medium is not supplemented with
yeast extract, than when the medium is supplemented with yeast extract.
In a particular embodiment, the medium is not supplemented with yeast
extract.

[0139] The invention also provides methods for the production of one or
more alcohols from a gaseous substrate comprising CO, the methods
comprising maintaining a culture of one or more bacterial isolate of the
invention in the presence of the gaseous substrate, and the anaerobic
fermentation of the gaseous substrate to one or more alcohols by the one
or more bacterial isolate.

[0140] The invention also provides a method for reducing the total
atmospheric carbon emissions from an industrial process, the method
comprising: [0141] (a) capturing CO-containing gas produced as a result
of the industrial process, before the gas is released into the
atmosphere; [0142] (b) the anaerobic fermentation of the CO-containing
gas to produce one or more alcohols by a culture containing one or more
bacterial isolates of the invention.

[0143] In certain embodiments of the methods of the invention, acetate is
produced as a by-product of the fermentation. The alcohol produced is
ethanol.

[0144] In certain embodiments, the culture is maintained in a liquid
nutrient medium.

[0145] The fermentation may be carried out in any suitable bioreactor,
such as a continuous stirred tank reactor (CTSR), a bubble column reactor
(BCR) or a trickle bed reactor (TBR). Also, in some preferred embodiments
of the invention, the bioreactor may comprise a first, growth reactor in
which the micro-organisms are cultured, and a second, fermentation
reactor, to which fermentation broth from the growth reactor is fed and
in which most of the fermentation product (ethanol and acetate) is
produced.

[0146] As described above, the carbon source for the fermentation reaction
is a gaseous substrate containing CO. The gaseous substrate may be a
CO-containing waste gas obtained as a by-product of an industrial
process, or from some other source such as from automobile exhaust fumes.
In certain embodiments, the industrial process is selected from the group
consisting of ferrous metal products manufacturing, such as a steel mill,
non-ferrous products manufacturing, petroleum refining processes,
gasification of coal, electric power production, carbon black production,
ammonia production, methanol production and coke manufacturing. In these
embodiments, the CO-containing gas may be captured from the industrial
process before it is emitted into the atmosphere, using any convenient
method. Depending on the composition of the gaseous CO-containing
substrate, it may also be desirable to treat it to remove any undesired
impurities, such as dust particles before introducing it to the
fermentation. For example, the gaseous substrate may be filtered or
scrubbed using known methods.

[0147] In addition, it is often desirable to increase the CO concentration
of a substrate stream (or CO partial pressure in a gaseous substrate) and
thus increase the efficiency of fermentation reactions where CO is a
substrate. Increasing CO partial pressure in a gaseous substrate
increases CO mass transfer into a fermentation medium. The composition of
gas streams used to feed a fermentation reaction can have a significant
impact on the efficiency and/or costs of that reaction. For example, O2
may reduce the efficiency of an anaerobic fermentation process.
Processing of unwanted or unnecessary gases in stages of a fermentation
process before or after fermentation can increase the burden on such
stages (e.g. where the gas stream is compressed before entering a
bioreactor, unnecessary energy may be used to compress gases that are not
needed in the fermentation). Accordingly, it may be desirable to treat
substrate streams, particularly substrate streams derived from industrial
sources, to remove unwanted components and increase the concentration of
desirable components.

[0148] Substrate streams derived from an industrial source are typically
variable in composition. Furthermore, substrate streams derived from
industrial sources comprising high CO concentrations (such as at least
50% CO or at least 65%) often have a low H2 component (such as less than
20% or less than 10% or 0%). As such, it is particularly desirable that
micro-organisms are capable of producing products by anaerobic
fermentation of substrates comprising a range of CO and H2
concentrations, particularly high CO concentrations and low H2
concentrations. The inventors tested C. autoethanogenum (obtained from
DSMZ under accession number DSM 10061) and note it would not grow and
produce products on gaseous substrates comprising CO without an H2
component. However, the bacteria of the present invention have the
surprising ability to grow and produce products (ethanol and acetate) by
fermenting a substrate comprising CO (and no H2).

[0149] The presence of hydrogen in the substrate stream can lead to an
improvement in efficiency of overall carbon capture and/or ethanol
productivity. For example, WO02/08438 describes the production of ethanol
using gas stream of various compositions. WO02/08438 reports a substrate
stream comprising 63% H2, 32% CO and 5% CH4 being provided to a culture
of C. ljungdahlii in a bioreactor to promote microbial growth and ethanol
production. When the culture reached a steady state and microbial growth
was no longer the main objective, the substrate stream was switched to
15.8% H2, 36.5% CO, 38.4% N2 and 9.3% CO2 in order to provide CO in a
slight excess and promote ethanol production. This document also
describes gas streams with higher and lower CO and H2 concentrations.

[0150] It will be appreciated that the processes of the present invention
as described herein can be used to reduce the total atmospheric carbon
emissions from industrial processes, by capturing CO-containing gases
produced as a result of such processes and using them as substrates for
the fermentation processes described herein.

[0151] Alternatively, in other embodiments of the invention, the
CO-containing gaseous substrate may be sourced from the gasification of
biomass. The process of gasification involves partial combustion of
biomass in a restricted supply of air or oxygen. The resultant gas
typically comprises mainly CO and H2, with minimal volumes of
CO2, methane, ethylene and ethane. For example, biomass by-products
obtained during the extraction and processing of foodstuffs such as sugar
from sugarcane, or starch from maize or grains, or non-food biomass waste
generated by the forestry industry may be gasified to produce a
CO-containing gas suitable for use in the present invention.

[0152] It is generally preferred that the CO-containing gaseous substrate
contains a major proportion of CO. In particular embodiments, the gaseous
substrate comprises at least about 65%, or at least about 70% to about
95% CO by volume. It is not necessary for the gaseous substrate to
contain any hydrogen. The gaseous substrate also optionally contains some
CO2, such as about 1% to about 30% by volume, such as about 5% to
about 10% CO2.

[0153] It will be appreciated that for growth of the bacteria and
CO-to-ethanol fermentation to occur, in addition to the CO-containing
substrate gas, a suitable liquid nutrient medium will need to be fed to
the bioreactor. A nutrient medium will contain vitamins and minerals
sufficient to permit growth of the micro-organism used. Anaerobic media
suitable for the fermentation of ethanol using CO as the sole carbon
source are known in the art. For example, suitable media are described in
U.S. Pat. Nos. 5,173,429 and 5,593,886 and WO 02/08438 as well as other
publications referred to herein before. In one embodiment of the
invention the media is LM23 as described in the Examples herein after.

[0154] The fermentation should desirably be carried out under appropriate
conditions for the CO-to-ethanol fermentation to occur. Reaction
conditions that should be considered include pressure, temperature, gas
flow rate, liquid flow rate, media pH, media redox potential, agitation
rate (if using a continuous stirred tank reactor), inoculum level,
maximum gas substrate concentrations to ensure that CO in the liquid
phase does not become limiting, and maximum product concentrations to
avoid product inhibition.

[0155] The optimum reaction conditions will depend partly on the
particular micro-organism of the invention used. However, in general, it
is preferred that the fermentation be performed at pressure higher than
ambient pressure. Operating at increased pressures allows a significant
increase in the rate of CO transfer from the gas phase to the liquid
phase where it can be taken up by the micro-organism as a carbon source
for the production of ethanol. This in turn means that the retention time
(defined as the liquid volume in the bioreactor divided by the input gas
flow rate) can be reduced when bioreactors are maintained at elevated
pressure rather than atmospheric pressure.

[0156] Also, since a given CO-to-ethanol conversion rate is in part a
function of the substrate retention time, and achieving a desired
retention time in turn dictates the required volume of a bioreactor, the
use of pressurized systems can greatly reduce the volume of the
bioreactor required, and consequently the capital cost of the
fermentation equipment. According to examples given in U.S. Pat. No.
5,593,886, reactor volume can be reduced in linear proportion to
increases in reactor operating pressure, i.e. bioreactors operated at 10
atmospheres of pressure need only be one tenth the volume of those
operated at 1 atmosphere of pressure.

[0157] The benefits of conducting a gas-to-ethanol fermentation at
elevated pressures have also been described elsewhere. For example, WO
02/08438 describes gas-to-ethanol fermentations performed under pressures
of 30 psig and 75 psig, giving ethanol productivities of 150 g/l/day and
369 g/l/day respectively. However, example fermentations performed using
similar medium and input gas compositions at atmospheric pressure were
found to produce between 10 and 20 times less ethanol per litre per day.

[0158] It is also desirable that the rate of introduction of the
CO-containing gaseous substrate is such as to ensure that the
concentration of CO in the liquid phase does not become limiting. This is
because a consequence of CO-limited conditions may be that the ethanol
product is consumed by the culture.

[0159] In certain embodiments, a fermentation process according to the
present invention described above will result in a fermentation broth
comprising ethanol, as well as bacterial cells, in the aqueous culture
medium. In preferred embodiments of the method the ethanol is recovered
from the fermentation broth.

[0160] In certain embodiments, the recovering of ethanol comprises
continuously removing a portion of broth and recovering the alcohol from
the removed portion of the broth.

[0161] In particular embodiments the recovery of ethanol includes passing
the removed portion of the broth containing ethanol through a separation
unit to separate bacterial cells from the broth, to produce a cell-free
alcohol-containing permeate, and returning the bacterial cells to the
bioreactor.

[0162] In certain embodiments, the methods of the invention are continuous
processes.

[0163] In particular embodiments acetate is produced as a by-product of
the fermentation.

[0164] In a further embodiment the ethanol and the acetate are recovered
from the broth.

[0165] In certain embodiments, the recovering of ethanol and acetate
comprises continuously removing a portion of the broth and recovering
separately ethanol and acetate from the removed portion of the broth.

[0166] In some embodiments the recovery of ethanol and acetate includes
passing the removed portion of the broth containing ethanol and acetate
through a separation unit to separate bacterial cells from the ethanol
and acetate, to produce a cell-free ethanol- and acetate-containing
permeate, and returning the bacterial cells to the bioreactor.

[0167] In the above embodiments, the recovery of ethanol and acetate
preferably includes first removing ethanol from the cell-free permeate
followed by removing acetate from the cell-free permeate. Preferably the
cell-free permeate is then returned to the bioreactor.

[0168] In certain embodiments, the methods of the invention are continuous
processes.

[0169] Ethanol is the preferred desired end product of the fermentation.
The ethanol may be recovered from the fermentation broth by methods known
in the art, such as fractional distillation or evaporation, and
extractive fermentation. Distillation of ethanol from a fermentation
broth yields an azeotropic mixture of ethanol and water (i.e. 95% ethanol
and 5% water). Anhydrous ethanol can subsequently be obtained through the
use of molecular sieve ethanol dehydration technology, which is also well
known in the art. Extractive fermentation procedures involve the use of a
water-miscible solvent that presents a low toxicity risk to the
fermentation organism, to recover the ethanol from the dilute
fermentation broth. For example, oleyl alcohol is a solvent that may be
used in this type of extraction process. Oleyl alcohol is continuously
introduced into a fermenter, whereupon this solvent rises forming a layer
at the top of the fermenter which is continuously extracted and fed
through a centrifuge. Water and cells are then readily separated from the
oleyl alcohol and returned to the fermenter while the ethanol-laden
solvent is fed into a flash vaporization unit. Most of the ethanol is
vaporized and condensed while the oleyl alcohol is non volatile and is
recovered for re-use in the fermentation.

[0170] Acetate may also be recovered from the fermentation broth using
methods known in the art. Methods for the recovery of acetate are
described in detail in WO2007/117157 and WO2008/115080.

[0171] In certain embodiments of the invention, ethanol and acetate are
recovered from the fermentation broth by continuously removing a portion
of the broth from the fermentation bioreactor, separating microbial cells
from the broth (conveniently by filtration), and recovering first ethanol
and then acetate from the broth. The ethanol may conveniently be
recovered by distillation, and the acetate may be recovered by adsorption
on activated charcoal, using the methods described above. The separated
microbial cells are preferably returned to the fermentation bioreactor.
The cell free permeate remaining after the ethanol and acetate have been
removed is also preferably returned to the fermentation bioreactor.
Additional nutrients (such as B vitamins) may be added to the cell free
permeate to replenish the nutrient medium before it is returned to the
bioreactor. Also, if the pH of the broth was adjusted as described above
to enhance adsorption of acetic acid to the activated charcoal, the pH
should be re-adjusted to a similar pH to that of the broth in the
fermentation bioreactor, before being returned to the bioreactor.

Reaction Stoichiometry

[0172] Without wishing to be bound by any theory, the chemical reactions
for the fermentation of CO to ethanol (a) and acetic acid (b) in the
process of the present invention are believed to be as follows:

18CO+9H2O=>3CH3CH2OH+12CO2 (a)

12CO+6H2O=>3CH3COOH+6CO2 (b)

The invention will now be described in more detail with reference to the
following non-limiting examples.

EXAMPLES

Media

[0173] The composition of media components used in the following examples
is provided in Tables 1 and 2.

[0174] LM17, LM23 and LM 33 media were prepared at pH 5.5 as follows. All
ingredients with the exception of cysteine HCL were mixed in dH2O to
a total volume of 1 L. This solution was made anaerobic by heating to
boiling and allowing it to cool to room temperature under a constant flow
of 95% CO, 5% CO2 gas. Once cool, the cysteine HCL was added and the
pH of the solution adjusted to 5.5; anaerobicity was maintained
throughout the experiments.

[0176] The Gas Chromatograph was operated in Split mode with a total flow
of hydrogen of 50 mL/min with 5 mL purge flow (1:10 split), a column head
pressure of 20 psig resulting in a linear velocity of 45 cm/sec. The
temperature program was initiated at 60° C., hold for 1 minute
then ramped to 170° C. at 30° C. per minute. This resulted
in a total run time of 4.65 minutes. Injector temperature was 180°
C. and the detector temperature was 225° C.

[0178] To determine the cell density in these experiments, the absorbance
of the samples was measured at 600 nm (spectrophotometer) and the dry
mass determined via calculation according to published procedures. The
level of metabolites was characterized using High Performance Liquid
Chromatography (HPLC) and in some cases Gas Chromatography (GC).

[0180] The method of sample preparation for HPLC was as follows: 400 μL
of sample and 50 μL of 0.15M ZnSO4 and 50 μL of 0.15M
Ba(OH)2 are loaded into an Eppendorf tube. The tubes are centrifuged
for 10 min. at 12,000 rpm, 4° C. 200 μL of the supernatant are
transferred into an HPLC vial, and 5 μL are injected into the HPLC
instrument.

Example 1

Production of a New Bacterial Isolate of the Invention

[0181] The new strain Clostridium autoethanogenum LBS1560 was produced
through a dedicated program of selection and propagation of microbial
cultures initiated from the parent C. autoethanogenum culture (DSMZ
10061) over a period of 18 months.

Methods

[0182] A frozen stock of C. autoethanogenum 10061 (obtained from DSMZ) was
initially thawed and used to inoculate LM23 medium prepared with 5
g/litre yeast extract in the presence of 95% CO and 5% CO2. This
culture could not be made to grow on LM23 medium in the absence of Yeast
Extract. In an effort to overcome the cultures dependence on yeast
extract over a period of months, actively growing microbial cultures that
were observed to produce the most ethanol and the highest ratio of
ethanol to acetate were repeatedly subcultured into medium containing
ever decreasing concentrations of yeast extract, always in the presence
of 95% CO 5% CO2 headspace gas. After this period cultures growing
and producing ethanol and acetate in the absence of yeast extract could
be observed. This selection protocol was actively maintained to further
identify and select for cultures that:

i) grew most rapidly; ii) produced the most ethanol; iii) produced the
highest ratio of ethanol to acetate; and, iv) grew in the absence of
yeast extract in the liquid medium.

Example 1.1

Rapid Growth Selection

[0183] In order to select for fast growing cultures, the micro-organisms
propensity to produce acetic acid as a by-product of energy metabolism
during periods of growth on a continuous 95% CO, 5% CO2 gas stream was
exploited. The accumulation of acetic acid in the growth medium has the
effect of lowering the pH of the process. Accordingly, a fermenter
configuration that diluted the culture in a growth dependent way in order
to introduce a pressure that would select for the fastest growing
populations was developed. An exemplary configuration is shown in FIG. 1,
wherein a culture of micro-organisms was fermented in a bioreactor 1. pH
of the nutrient media 2 was monitored by a conventional pH probe 3.
Deviations in the pH reading from the set point of 5.5 caused a pump 4 to
be activated, however, rather than the signal from the probe being
relayed to a pump that dosed a base or acid solution; in this case the
pump was linked to a bottle 5 containing fresh anaerobic LM17 medium at
pH 5.8. Thus as the culture grew, acetic acid was produced, the pH of the
medium 2 began to drop causing the activation of a pump 4 that introduced
medium at pH 5.8. The pump 4 was only de-activated once the medium pH was
returned to 5.5 or above. The liquid level in the reactor 1 was
maintained using a level probe 6 linked to a second pump 7 that operated
to maintain the liquid level in the bioreactor 1 at or below a fixed
level. Medium pumped away from bioreactor 1 was passed to waste
container/means 8. Accordingly, the growing culture population was
diluted in a growth-linked manner and the faster the population grew, the
more acetate was produced and more fresh medium was introduced until
ultimately, relatively large volumes of medium were introduced into the
fermenter to maintain pH effectively selecting for the fastest growing
populations as these would not be washed out in an effort to maintain the
liquid volume of the vessel at a fixed level. This fermenter
configuration was maintained for several months at a time as a continuous
culture in order to isolate fast growing cultures. Every 14 days, an
aliquot of the culture was removed and allowed to grow in a 250 ml serum
bottle containing 50 ml of medium and 35 psig of 95% CO, 5% CO2 in the
headspace. Once actively growing the culture was prepared and stored as a
glycerol stock for comparative work with the original culture stocks.

Results

[0184] The process of selection and subculture over a period of 18 months
described above resulted in the new strain LBS1560 which showed optimum
performance for each of features i) to iv) above. The new strain of
bacteria was observed to be Gram positive (it stained Gram positive),
non-motile, having a rod shape, and surprisingly exhibiting little to no
ability to sporulate (as described further herein after).

[0185] LBS1560 was deposited at the DSMZ, Germany, in accordance with the
Budapest Treaty, on 19 Oct. 2007, and allocated the accession number DSM
19630.

Example 2

Culture and Storage of LBS1560

[0186] C. autoethanogenum LBS1560 can be cultivated using the following
conditions: growth on 95% CO gas (5% CO2) 35 psi in LM23 medium, at
37° C., pH 5.5, with agitation (200 rpm shaking) under anaerobic
conditions. Growth may be monitored by measuring OD at 600 nm and
microscopic analysis.

[0187] For storage, a log phase culture of LBS1560 in LM23 +20% glycerol
is flash frozen and then stored at -80° C.

Example 3

Comparison of the New C. autoethanogenum LBS1560 with the Original
Parental Strain C. autoethanogenum DSMZ 10061

[0188] This experiment demonstrates the improved efficiency of the new
strain LBS1560 for the anaerobic fermentation of a CO-containing gaseous
substrate into ethanol, in comparison with the parental strain C.
autoethanogenum DSMZ 10061. This experiment also demonstrates efficient
fermentation of CO-containing gas to ethanol by the new strain LBS1560 in
the presence of high levels CO and in the absence of H2.

Methods

[0189] Frozen stocks of the selected microbial culture LBS1560, and the
original parent culture DSMZ 10061 were taken, thawed and used to
inoculate sealed 15 ml Hungate tubes containing 5 ml of minimal liquid
anaerobic microbial growth medium (LM23) either in the presence or
absence of 0.1% (w/v) yeast extract (YE). All Hungate tubes were
maintained under a 95% CO, 5% CO2 gas atmosphere. For each Hungate
tube, microbial growth, ethanol and acetate production were monitored
over a 7 day culture period.

[0191] The data presented in Table 3 highlight several reproducible
differences between strain LBS1560 and the parent strain DSMZ 10061. DSMZ
10061 was unable to grow in minimal medium that lacked yeast extract,
while LBS1560 could grow in medium in the presence or absence of yeast
extract, but performed best on minimal medium that lacked yeast extract.
LBS1560 grown on minimal medium performed better in terms of growth,
ethanol production, and ethanol to acetate ratio than DSMZ 10061 grown on
medium containing yeast extract.

Example 4

Sporulation Characteristics of LBS1560

[0192] To identify sporulation characteristics, LBS1560 was exposed to
various conditions known to induce spore formation in bacteria in
accordance with the methodology detailed below.

[0193] Starvation: a culture of LBS1560 was suspended in sterile distilled
water

[0194] Exposure to Oxygen: sterile air was injected into the head space of
Hungate tube containing 5 ml of growing culture, then the tube was placed
on shaker and incubated at 37° C.

[0195] Exposure to low pH medium (pH 3): microbes were grown in LM23 (pH
5.5) to a high cell concentration, then the medium was exchanged to fresh
growth medium pH 3.

[0196] Exposure to Oxygen and Fructose as carbon and energy source: liquid
medium contained 5 g/L of fructose and no reducing agent (i.e.
cysteine-HCl) was saturated with oxygen and a high concentration of cells
were suspended in this medium for 2 days.

[0197] The ability of LBS1560 to sporulate was determined by microscopic
examination. Bacterial samples were stained with coomassie blue which
facilitates the observation of spores. LBS1560 were observed on a number
of occasions. Essentially none of the bacterial population were observed
to exhibit spores. It was noted that while isolated spores were observed
by microscopy in some instances, they were estimated to be significantly
less than 0.1% of the overall microbial population. This was surprising
and unexpected given that the parent strain and related strains of
Clostridia are known to sporulate. The inability to sporulate provides
advantages to the bacteria of the invention as herein before described.

Example 5

Ethanol Production by LBS1560

[0198] This example describes continuous ethanol production by LBS1560
over an extended period. FIG. 2 provides a summary of the concentrations
of acetate, ethanol and biomass over a 2 week period.

Procedure

[0199] 1. 1 L media of anaerobic LM33 fermentation media in a 1 Litre CSTR
was inoculated with an actively growing Clostridium autoethanogenum
(LBS1560) culture (DSMZ 19630) at a level of 5% (v/v). A continuous flow
of 70% CO and 15% CO2 1% H2 14% N2 gas was introduced at
the bottom of the fermenter vessel through a diffusing sparger at a
volumetric flow rate of 19 ml/minutes. The initial pH of the fermenter
was set to 5.5 and the agitation speed was adjusted to 400 rpm. 2. For
the majority of the experiment, the acetic acid concentration of the
culture was maintained below 4 g/L by a cell recycle and media exchange
system. The cells were passed through a cross flow membrane Viva 200, the
filtrate was collected and the cells were returned to the reactor vessel.
The filtrate was replaced with fresh media to ensure the medium volume
inside the reactor remained constant. 3. The culture was operated
continuously for at least 14 days. The cell recycle system removed 1-1.5
L of liquid nutrient media every 1-2 days without removing bacteria from
the bioreactor. The removed media was replaced with fresh media, to
maintain constant volume. 4. The pH of the fermenter was increased from
5.6 to 6.0 over the first four days of the experiment.

Results

[0200] The rapid growth phase of acetogenic bacteria (such as C.
autoethanogenum) is typically associated with high acetate production in
a controlled fermentation environment. In this experiment, using the
novel strain LBS1560, during the growth phase (day 0-3) the culture
produced an average of 0.3 g/L/day acetate and 0.16 g/L/day ethanol.
Following the growth phase (day 3-13) the culture produced an average of
1.03 g/L/day acetate and an average of 1.4 g/L/day ethanol. Over the
alcohol production period total ethanol produced was 14 g/L. The results
show a lower than expected level of acetate production and significantly
higher ethanol production.

[0201] The specific methods and compositions described herein are
representative of preferred embodiments and are exemplary and not
intended as limitations on the scope of the invention. Other objects,
aspects and embodiments will occur to those skilled in the art upon
consideration of this specification, and are encompassed within the scope
and spirit of the invention. It will be readily apparent to one skilled
in the art that varying substitutions and modifications may be made to
the invention disclosed herein without departing from the scope and
spirit of the invention. The invention illustratively described herein
suitably may be practised in the absence of any element or elements, or
limitation or limitations, which is not specifically disclosed herein as
essential. Thus, for example, in each instance herein, in embodiments or
examples of the present invention, the terms "comprising", "including",
"containing" etc are to be read expansively and without limitation.
Furthermore, titles, headings, or the like are provided to enhance the
reader's comprehension of this document, and should not be read as
limiting the scope of the present invention.

[0202] The entire disclosures of all applications, patents and
publications, cited above and below, if any, are hereby incorporated by
reference. However, the reference to any applications, patents and
publications in this specification is not, and should not be taken as, an
acknowledgment or any form of suggestion that they constitute valid prior
art or form part of the common general knowledge in any country in the
world.